The aim of this study is to investigate alterations in cell state which affect cellular radiosensitivity and are inducible by environmental deprivation of the kind seen in tumors, i.e. oxygen/nutrient deprivation or reperfusion following deprivation. The analysis of changes affecting sensitivity is divided into three parts: a) quantitative assessment of changes at the DNA level and the radioresponse of the cells exhibiting modified DNA; b) demonstration and measurement of fundamental, dynamic alterations in cellular activities (e.g. differentiation, drug uptake); c) detection of intrinsic subcellular markers indicative of previous cellular deprivation and of a modified phenotype with altered radiosensitivity. The sandwich system (in vitro cell-culture) will be used to experimentally model tumor-like populations of cells having nutrient and metabolite gradients arising due to distance from vascularization. In sandwiches, as in tumors, imposed gradients in environmental condition induce gradients in cellular phenotype and cellular radioresponse. Many of the detection assays to be used take advantage of the ability to make in situ measurements on the single cell level and not lose information on the spatial location of cells within the population. The geometry of the sandwich system induces population heterogeneity to develop in a spacially organized manner. Thus, sorting out of cause and effect becomes achievable. Interactions between subpopulations of cells of differing character exist, yet due to the monolayer nature, complete cell access and visibility are maintained, permitting assessment of both individual cell sensitivity and whole population response. The proposed work has experimental and theoretical components. Initial studies present novel biological results revealed by our gradient approach to deprivation-induced phenomena (e.g. alterations in DNA content and sub-nuclear distribution, induction of differentiation, increased mitochondrial membrane potential).
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